Human 3-hydroxy-3-methylglutaryl coenzyme A reductase. Conserved domains responsible for catalytic activity and sterol-regulated degradation.

Self Cite

118

Abstract. 3-Hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase is anchored to the endoplasmic reticulum (ER) membrane by a hydrophobic NH2-terminal domain that contains seven apparent membrane-spanning regions and a single N-linked carbohydrate chain. The catalytic domain, which includes the COOH-terminal two-thirds of the protein, extends into the cytoplasm. The enzyme is normally degraded with a rapid half-life (2 h), but when cells are depleted of cholesterol, its half-life is prolonged to 11 h. Addition of sterols accelerates degradation by fivefold. To explore the requirements for regulated degradation, we prepared expressible reductase cDNAs from which we either deleted two contiguous membrane-spanning regions (numbers 4 and 5) or abolished the single site for N-linked glycosylation. When expressed in hamster cells after transfection, both enzymes retained catalytic activity. The deletionbearing enzyme continued to be degraded with a rapid half-life in the presence of sterols, but it no longer was stabilized when sterols were depleted. The glycosylation-minus enzyme was degraded at a normal rate and was stabilized normally by sterol deprivation. When cells were induced to overexpress the deletionbearing enzyme, they did not incorporate it into neatly arranged crystalloid ER tubules, as occurred with the normal and carbohydrate-minus enzymes. Rather, the deletion-bearing enzyme was incorporated into hypertrophied but disordered sheets of ER membrane. We conclude that the carbohydrate component of HMG CoA reductase is not required for proper subcellular localization or regulated degradation. In contrast, the native structure of the transmembrane component is required to form a normal crystalloid ER and to allow the enzyme to undergo regulated degradation by sterols.

Section: Discussionmentioning

confidence: 99%

Partial deletion of membrane-bound domain of 3-hydroxy-3-methylglutaryl coenzyme A reductase eliminates sterol-enhanced degradation and prevents formation of crystalloid endoplasmic reticulum.

Jingami¹,

Brown²,

Goldstein³

et al. 1987

Self Cite

118

“…The NH2terminal of the yeast isozymes have been proposed to have seven transmembrane spans (Sengstag et al, 1990) whereas mammalian HMG-R appears to have eight (Olender and Simon, 1992;. Furthermore, although the yeast NH2-termini are similar to each other (>50% se-quence identity) they bear no sequence resemblance to mammalian NH2-terminal regions, which are conserved amongst themselves (Liscum et al, 1985;Luskey and Stevens, 1985) and some other metazoans (Chin et al, 1982;Woodward et al, 1988). The similarities between yeast and animal cell HMG-R, along with indications that yeast may regulate HMG-R levels in response to changes in isoprenoid synthesis, led us to investigate the degradation of yeast HMG-R, and whether the stability of this enzyme was regulated.…”

Section: Sterolsmentioning

confidence: 99%

“…Mammalian HMG-R has two distinct structural domains: a COOH-terminal catalytic region connected by a linker to an NH2-terminal region that anchors the enzyme to the ER membrane by virtue of its multiple membrane-spanning domains (Liscum et al, 1985;Luskey and Stevens, 1985). The NH2-terminal region is not required for catalysis, but is required for regulated ER degradation of the native enzyme (Nakanishi et al, 1988), or of fusion proteins bearing this region (Chun et al, 1990).…”

mentioning

confidence: 99%

Regulated degradation of HMG-CoA reductase, an integral membrane protein of the endoplasmic reticulum, in yeast.

Hampton

Rine

1994

209

264

Abstract. Numerous integral membrane proteins are degraded in the mammalian ER. HMG-CoA reductase (HMG-R), a key enzyme in the mevalonate pathway by which isoprenoids and sterols are synthesized, is one substrate of ER degradation. The degradation of HMG-R is modulated by feedback signals from the mevalonate pathway. We investigated the role of regulated degradation of the two isozymes of HMG-R, Hmglp and Hmg2p, in the physiology of Saccharomyces cerevisiae. Hmglp was quite stable, whereas Hmg2p was rapidly degraded. Degradation of Hmg2p proceeded independently of vacuolar proteases or secretory traffic, indicating that Hmg2p degradation occurred at the ER. Hmg2p stability was strongly affected by modulation of the mevalonate pathway through pharmacological or genetic means. Decreased mevalonate pathway flux resulted in decreased degradation of Hmg2p. One signal for degradation of Hmg2p was a nonsterol, mevalonate-derived molecule produced before the synthesis of squalene. Genetic evidence indicated that a farnesylated protein may also be necessary for Hmg2p degradation. Studies with reporter genes demonstrated that the stability of each isozyme was determined by its noncatalytic NH2-terminal domain. Our data show that ER protein degradation is widely conserved among eukaryotes, and that feedback control of HMG-R degradation is an ancient paradigm of regulation.

“…Through cloning of its cDNA, the primary structure of HMG-CoA reductase from many eukaryotes, including yeast (Basson et al, 1988), plants (Learned and Fink, 1989;Caelles et al, 1989), fly (Gertler et al, 1988), schistosomes (Rajkovic et al, 1989), sea urchin (Woodward et al, 1988), frog (Chen and Shapiro, 1990), rodents (Chin et al, 1984;Skalnik and Simoni, 1985), and humans (Luskey and Stevens, 1985) has been deduced. Mammalian HMG-CoA reductase is a 97-kD membrane-bound "high mannose" glycoprotein of the ER (Liscum et al, 1983b;Brown and Simoni, 1984).…”

mentioning

confidence: 99%

Immunological evidence for eight spans in the membrane domain of 3-hydroxy-3-methylglutaryl coenzyme A reductase: implications for enzyme degradation in the endoplasmic reticulum

Roitelman

Olender

Bar-Nun

et al. 1992

171

133

Abstract. We have raised two monospecific antibodies against synthetic peptides derived from the membrane domain of the ER glycoprotein 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, the rate limiting enzyme in the cholesterol biosynthetic pathway. This domain, which was proposed to span the ER membrane seven times (Liscum, L., J. FinerMoore, R. M. Stroud, K. L. Luskey, M. S. Brown, and J. L. Goldstein. 1985. J. Biol. Chem. 260:522-538), plays a critical role in the regulated degradation of the enzyme in the ER in response to sterols. The antibodies stain the ER of cells and immunoprecipitate HMG-CoA reductase and HMGal, a chimeric protein composed of the membrane domain of the reductase fused to Escherichia coli/3-galactosidase, the degradation of which is also accelerated by sterols. We show that the sequence Arg TM through Leu 242 of HMG-CoA reductase (peptide G) faces the cytoplasm both in cultured cells and in rat liver, whereas the sequence Thr TM through Glu m (peptide H) faces the lumen of the ER. This indicates that a sequence between peptide G and peptide H spans the membrane of the ER. Moreover, by epitope tagging with peptide H, we show that the loop segment connecting membrane spans 3 and 4 is sequestered in the lumen of the ER. These results demonstrate that the membrane domain of HMG-CoA reductase spans the ER eight times and are inconsistent with the seven membrane spans topological model. The approximate boundaries of the proposed additional transmembrane segment are between Lys 24s and Asp 276. Replacement of this 7th span in HMGal with the first transmembrane helix of bacteriorhodopsin abolishes the sterol-enhanced degradation of the protein, indicating its role in the regulated turnover of HMG-CoA reductase within the endoplasmic reticulum.3-HYDROXY-3-METHYLGLUTARYL coenzyme A (HMGCoA) t reductase catalyzes the conversion of HMG-CoA to mevalonate (Durr and Rudney, 1960), the precursor for a wide variety of metabolites that play central roles in cellular functions. These products of the mevalonate pathway include sterols, ubiquinone, dolichols, isopentenyladenine, heme A and the isoprenyl moieties of proteins (for a recent review see Goldstein and Brown, 1990). In mammalian cells, HMG-CoA reductase is the major regulatory enzyme in this pathway and is subject to complex metabolic control ensuring an adequate supply of intermediates and products of this pathway. This is achieved by regulation of transcription of the HMG-CoA reductase gene (Liscum et al., 1983a;Osborne et al., 1985), regulation of translation of its mRNA (Tanaka et al., 1983; Petfley and Sinensky, 1985; Nakanishi Shoshana Bar-Nun is a visiting Scholar from the